Atomic Force Microscope Operating Research Articles

Atomic Force Microscopy: An Introduction.

Imaging of nano-sized particles and sample features is crucial in a variety of research fields, for instance, in biological sciences, where it is paramount to investigate structures at the single particle level. Often, two-dimensional images are not sufficient, and further information such as topography and mechanical properties are required. Furthermore, to increase the biological relevance, it is desired to perform the imaging in close to physiological environments. Atomic force microscopy (AFM) meets these demands in an all-in-one instrument. It provides high-resolution images including surface height information leading to three-dimensional information on sample morphology. AFM can be operated both in air and in buffer solutions. Moreover, it has the capacity to determine protein and membrane material properties via the force spectroscopy mode. Here we discuss the principles of AFM operation and provide examples of how biomolecules can be studied. New developments in AFM are discussed, and by including approaches such as bimodal AFM and high-speed AFM (HS-AFM), we show how AFM can be used to study a variety of static and dynamic single biomolecules and biomolecular assemblies.

Focus on lasers, imaging, microscopy, and nanoscience

Focus on lasers, imaging, microscopy, and nanoscience

Hybrid mode atomic force microscopy of phase modulation and frequency modulation.

We propose hybrid phase modulation (PM)/frequency modulation (FM) atomic force microscopy (AFM) to increase the imaging speed of AFM in high-Q environments. We derive the relationship between the phase shift, the frequency shift and the tip-sample interaction force from the equation of motion for the cantilever in high-Q environments. The tip-sample conservative force is approximately given by the sum of the conservative force with respect to the phase shift in the PM mode and that with respect to the frequency shift in the FM mode. We preliminarily demonstrate that the hybrid PM/FM-AFM is a new and very promising AFM operation mode that can increase imaging speed.

A cantilever-based, ultrahigh-vacuum, low-temperature scanning probe instrument for multidimensional scanning force microscopy.

Cantilever-based atomic force microscopy (AFM) performed under ambient conditions has become an important tool to characterize new material systems as well as devices. Current instruments permit robust scanning over large areas, atomic-scale lateral resolution, and the characterization of various sample properties using multifrequency and multimodal AFM operation modes. Research of new quantum materials and devices, however, often requires low temperatures and ultrahigh vacuum (UHV) conditions and, more specifically, AFM instrumentation providing atomic resolution. For this, AFM instrumentation based on a tuning fork force sensor became increasingly popular. In comparison to microfabricated cantilevers, the more macroscopic tuning forks, however, lack sensitivity, which limits the measurement bandwidth. Moreover, multimodal and multifrequency techniques, such as those available in cantilever-based AFM carried out under ambient conditions, are challenging to implement. In this article, we describe a cantilever-based low-temperature UHV AFM setup that allows one to transfer the versatile AFM techniques developed for ambient conditions to UHV and low-temperature conditions. We demonstrate that such a cantilever-based AFM offers experimental flexibility by permitting multimodal or multifrequency operations with superior force derivative sensitivities and bandwidths. Our instrument has a sub-picometer gap stability and can simultaneously map not only vertical and lateral forces with atomic-scale resolution, but also perform rapid overview scans with the tip kept at larger tip–sample distances for robust imaging.

Deep Learning for Live Cell Shape Detection and Automated AFM Navigation.

Atomic force microscopy (AFM) provides a platform for high-resolution topographical imaging and the mechanical characterization of a wide range of samples, including live cells, proteins, and other biomolecules. AFM is also instrumental for measuring interaction forces and binding kinetics for protein–protein or receptor–ligand interactions on live cells at a single-molecule level. However, performing force measurements and high-resolution imaging with AFM and data analytics are time-consuming and require special skill sets and continuous human supervision. Recently, researchers have explored the applications of artificial intelligence (AI) and deep learning (DL) in the bioimaging field. However, the applications of AI to AFM operations for live-cell characterization are little-known. In this work, we implemented a DL framework to perform automatic sample selection based on the cell shape for AFM probe navigation during AFM biomechanical mapping. We also established a closed-loop scanner trajectory control for measuring multiple cell samples at high speed for automated navigation. With this, we achieved a speed-up in AFM navigation and reduced the time involved in searching for the particular cell shape in a large sample. Our innovation directly applies to many bio-AFM applications with AI-guided intelligent automation through image data analysis together with smart navigation.

On the Possibility of Using Glass Capillaries as Probes of an Atomic-Force Microscope Operating in a Hybrid Mode

On the Possibility of Using Glass Capillaries as Probes of an Atomic-Force Microscope Operating in a Hybrid Mode

Correlative nanoscopy: A multimodal approach to molecular resolution.

Atomic force microscopy (AFM) is a nano‐mechanical tool uniquely suited for biological studies at the molecular scale. AFM operation is based on mechanical interaction between the tip and the sample, a mechanism of contrast capable of measuring different information, including surface topography, mechanical, and electrical properties. However, the lack of specificity highlights the need to integrate AFM data with other techniques providing compositional hints. In particular, optical microscopes based on fluorescence as a mechanism of contrast can access the local distribution of specific molecular species. The coupling between AFM and super‐resolved fluorescence microscopy solves the resolution mismatch between AFM and conventional fluorescence optical microscopy. Recent advances showed that also the inherently label‐free imaging capabilities of the AFM are fundamental to complement the fluorescence images. In this review, we have presented a brief historical view on correlative microscopy, and, finally, we have summarized the progress of correlative AFM‐super‐resolution microscopy in biological research.

Scanning Probe Microscopies: Imaging and Biomechanics in Reproductive Medicine Research.

Basic and translational research in reproductive medicine can provide new insights with the application of scanning probe microscopies, such as atomic force microscopy (AFM) and scanning near-field optical microscopy (SNOM). These microscopies, which provide images with spatial resolution well beyond the optical resolution limit, enable users to achieve detailed descriptions of cell topography, inner cellular structure organization, and arrangements of single or cluster membrane proteins. A peculiar characteristic of AFM operating in force spectroscopy mode is its inherent ability to measure the interaction forces between single proteins or cells, and to quantify the mechanical properties (i.e., elasticity, viscoelasticity, and viscosity) of cells and tissues. The knowledge of the cell ultrastructure, the macromolecule organization, the protein dynamics, the investigation of biological interaction forces, and the quantification of biomechanical features can be essential clues for identifying the molecular mechanisms that govern responses in living cells. This review highlights the main findings achieved by the use of AFM and SNOM in assisted reproductive research, such as the description of gamete morphology; the quantification of mechanical properties of gametes; the role of forces in embryo development; the significance of investigating single-molecule interaction forces; the characterization of disorders of the reproductive system; and the visualization of molecular organization. New perspectives of analysis opened up by applying these techniques and the translational impacts on reproductive medicine are discussed.

Multi-Channel Exploration of O Adatom on TiO2(110) Surface by Scanning Probe Microscopy.

We studied the O2 dissociated state under the different O2 exposed temperatures with atomic resolution by scanning probe microscopy (SPM) and imaged the O adatom by simultaneous atomic force microscopy (AFM)/scanning tunneling microscopy (STM). The effect of AFM operation mode on O adatom contrast was investigated, and the interaction of O adatom and the subsurface defect was observed by AFM/STM. Multi-channel exploration was performed to investigate the charge transfer between the adsorbed O and the TiO2(110) by obtaining the frequency shift, tunneling current and local contact potential difference at an atomic scale. The tunneling current image showed the difference of the tunneling possibility on the single O adatom and paired O adatoms, and the local contact potential difference distribution of the O-TiO2(110) surface institutively revealed the charge transfer from TiO2(110) surface to O adatom. The experimental results are expected to be helpful in investigating surface/interface properties by SPM.

A horizontal-type scanning near-field optical microscope with torsional mode operation toward high-resolution and non-destructive imaging of soft materials.

We design and build a horizontal-type aperture based scanning near-field optical microscope (a-SNOM) with superior mechanical stability toward high-resolution and non-destructive topographic and optical imaging. We adopt the torsional mode in AFM (atomic force microscopy) operation to achieve a better force sensitivity and a higher topographic resolution when using pyramidal a-SNOM tips. The performance and stability of the AFM are evaluated through single-walled carbon nanotube and poly(3-hexyl-thiophene) nanowire samples. An optical resolution of 93 nm is deduced from the a-SNOM imaging of a metallic grating. Finally, a-SNOM fluorescence imaging of soft lipid domains is successfully achieved without sample damage by our horizontal-type a-SNOM instrument with torsional mode AFM operation.

Effects of the polysaccharide SPS-3-1 purified from Spirulina on barrier integrity and proliferation of Caco-2 cells

Following ultrasonication combined with a hot water extraction, a new type of bioactive polysaccharide, SPS-3-1, was purified from Spirulina using ultrafiltration centrifugation and gel filtration chromatography. The structure of SPS-3-1 was determined with high performance gel permeation chromatography, gas chromatography, periodate oxidation, fourier transform infrared spectroscopy, nuclear magnetic resonance, and atomic force microscopy performance. SPS-3-1 is a homogeneous β-pyran polysaccharide with 1 → 2, 1 → 3, and 1 → 4 glycosyl bonds, mainly composed of d-ribose, l-rhamnose, l-arabinose, l-foucose, and d-glucose. The molar ratio of these components is 1:0.70:1.03:2.1:6.59. The molecular weight of SPS-3-1 is 623.02 kDa. SPS-3-1 has a linear filament structure with a width of 34.132 nm and a height of 819.169 pm. We found that SPS-3-1 significantly enhanced transepithelial electrical resistance, a tight junction integrity marker, in a Caco-2 intestinal cell monolayer model. Analysis of the effect of SPS-3-1 on cell proliferation showed that SPS-3-1 inhibited the in vitro growth of Caco-2 and HepG2 cells with an IC50 of 566.67 μg/mL and 1078.95 μg/mL, respectively.

Atomic force microscopy as an imaging tool to study the bio/nonbio complexes.

Atomic force microscopy (AFM) besides X-ray crystallography and electron microscopy is one of the most attractive methods to study bio/nonbio complexes. Information on how biomacromolecules interact with nanomaterials under different environmental conditions has important implications for the practice of nanomedicine and concerning the safety of nanomaterials. These complexes cover a broad range both in terms of stability and composition. AFM offers a wealth of structural and functional data about such assemblies. The variety of samples investigated using AFM in biology includes nanometre-sized proteins, lipids, DNA, amyloid fibrils, as well as larger objects such as cells. Herein we choose to review the significance of AFM to study various biological aspects of selected assemblies. We have focused on the exploitation of AFM operating in the air. The presented AFM research offers a unique and often unexpected insight into the structure and function of the bio/nonbio complexes. LAY DESCRIPTION: Atomic force microscopy (AFM) besides X-ray crystallography and electron microscopy is one of the most attractive methods to study bio/nonbio complexes. Information on how biomacromolecules interact with nanomaterials under different environmental conditions has important implications for the practice of nanomedicine and concerning the safety of nanomaterials. These complexes cover a broad range both in terms of stability and composition. AFM offers a wealth of structural and functional data about such assemblies. The variety of samples investigated using AFM in biology includes nanometre-sized proteins, lipids, DNA, amyloid fibrils, as well as larger objects such as cells. Herein we choose to review the significance of AFM to study various biological aspects of selected assemblies. The presented AFM research offers a unique and often unexpected insight into the structure and function of the bio/nonbio complexes. Nature has set us a perfect example of how to elegantly optimise and fine tune different types of processes. The relatively young field of nanotechnology has studied biological processes and exploited their unique strengths as novel materials. The resulting area of bionanotechnology has adopted interaction schemes presented to us by biology, to provide enhanced selectivity, efficiency or versatility of molecular attachment strategies. Two scenarios of this synergistic scheme are: the conjugation of nanostructures as a tool for research in biological science and the conjugation of biological particles as a tool for nanotechnology. The use of nanotechnologies for medical applications raises high expectations regarding diagnosis, drug delivery, gene therapy, and tissue engineering. There is an increasing number of reports using AFM as a nanodiagnostic tool with patient cells. The use of AFM, in combination with more conventional analytical approaches, could inform decisions related to recommendations for treatments. Applying AFM techniques in nanomedicine is becoming well established. Atomic force microscopy (AFM) is one of the most functional and powerful microscopy technology for studying biological and material samples at the nanoscale. It is advantageous because an atomic force microscope can image three-dimensional topography of very small objects. It also provides various types of surface measurements to the needs of scientists and engineers if combined with other electromagnetic waves. It is powerful because an AFM can generate images at atomic resolution with 10-9 m scale resolution height information, with minimum sample preparation. AFM gives details on how biological molecules, such as nucleic acids, proteins, and amyloid aggregates, interact with nanomaterials under different environmental conditions. Here, we have shown several examples of relevant applications of AFM to study structural, functional and mechanical properties useful for the medicine and concerning the safety of nanomaterials.

Analysis of Dynamic Behavior of V-Shaped Atomic Force Microscope Cantilever in Contact and Tapping Modes by Considering Various Immersion Surroundings

In atomic force microscope (AFM) as a powerful device for scanning the samples, a cantilever is used to sense the variations of its dynamic characteristics due to the tip–sample interaction. Theoretical models that can accurately simulate the surface-coupled dynamics of the cantilever are necessary for quantifiable and qualitative explanation and understanding of measured results. A good understanding of the dynamics of AFM cantilevers vibrating in liquid is needed for the interpretation of scanning images, selection of AFM operating conditions and evaluation of sample’s mechanical properties. In this paper, the amplitude of frequency response functions (FRF) of vertical and rotational movements for a V-shaped atomic force microscope beam immersed in various surroundings has been surveyed for the first time. For rising the precision of theoretical model, we have considered all needful details for the beam and sample. The amplitude of FRF of vertical and rotational movements and resonant frequency of beam have been surveyed by supposing cantilever thickness, breadth and length, the angle between cantilever and sample surface, tip height and normal and lateral tip–sample interaction force. In this study, carbon tetrachloride (CCL4), methanol, acetone, water and air have been considered as surroundings. The results show that an increase in the tip–sample interaction force considerably raises the amount of resonant frequency. By increasing the liquid viscosity, the amplitude of FRF for vertical and rotational movements and resonant frequency reduce. Moreover, the amplitude of FRF of vertical and rotational movements is reduced by raising the rectangular and tapered parts lengths, but increased by raising the rectangular part breadth and cantilever thickness. The resonant frequency goes down by increasing the rectangular and tapered parts lengths and rectangular part breadth, but increases by rising the cantilever thickness. In this paper, we have tried to model the realistic dynamic behavior of V-shaped AFM beam in contact and tapping modes with the most accuracy. Results show good agreement. Based on the experimental results, the present theoretical model can forestall better the realistic behavior of tapping mode than the contact mode for V-shaped AFM cantilever. Based on the complexity of the V-shaped AFM cantilever, this study has been done for the first time in both of the contact and tapping modes.

Wear comparison of critical dimension-atomic force microscopy tips.

Nanoscale wear affects the performance of atomic force microscopy (AFM)-based measurements for all applications including process control measurements and nanoelectronics characterization. As such, methods to prevent or reduce AFM tip wear is an area of active research. However, most prior work has been on conventional AFMs rather than critical dimension AFM (CD-AFM). Hence, less is known about CD-AFM tip-wear. Given that tip-wear directly affects the accuracy of dimensional measurements, a basic understanding of CD-AFM tip wear is needed. Toward this goal, we evaluated the wear performance of electron beam deposited CD-AFM tips. Using a continuous scanning strategy, we evaluated the overall wear rate and tip lifetime and compared these with those of silicon-based CD-AFM tips. Our data show improved tip lifetime of as much as a factor of five and reduced wear rates of more than 17 times. Such improvements in wear rate means less measurement variability and lower cost.

Euler–Bernoulli theory accurately predicts atomic force microscope cantilever shape during non-equilibrium snap-to-contact motion

We prove that the Euler–Bernoulli elastic beam theory can be reliably used to describe the dynamics of an atomic force microscope cantilever during the far from equilibrium snap-to-contact event. In conventional atomic force microscope operation, force-separation curves are obtained by post-processing voltage versus time traces produced by measuring one point on the cantilever close to the hanging end. In this article, we assess the validity of the Euler–Bernoulli equation during the snap-to-contact event. The assessment is based on a direct comparison between experiment and theory. The experiment uses Doppler vibrometry to measure displacement versus time for many points along the long axis of the cantilever. The theoretical algorithm is based on a solution of the Euler–Bernoulli equation to obtain the full shape of the cantilever as a function of time. The algorithm uses as boundary conditions, experimentally obtained information only near the hanging end of the cantilever. The solution is obtained in a manner that takes into account non-equilibrium motion. Within experimental error, the theory agrees with experiment indicating that the Euler–Bernoulli theory is appropriate to predict the cantilever kinematics during snap-to-contact. Since forces on the tip can be obtained from the instantaneous shape of the cantilever, this work should allow for computation of tip-sample forces during the snap-to-contact event from a conventional force-distance measured input.

Rapid Probe Engagement and Withdrawal With Force Minimization in Atomic Force Microscopy: A Learning-Based Online-Searching Approach

In this article, the problem of rapid probe engagement and withdrawal in atomic force microscopy (AFM) is addressed. Probe engagement and withdrawal is needed in almost all AFM operations, ranging from imaging to nanomanipulation. However, due to the highly nonlinear force-distance relation, large probe-sample interaction force can be induced during the probe engagement and withdrawal process, resulting in sample deformation and damage and measurement errors. Rapid probe engagement and withdrawal is needed to achieve high-speed AFM operations, particularly, to capture and interrogate dynamic evolutions of the sample. We propose an online-searching-based optimization approach to minimize both the engagement (and withdrawal) time and the interaction force. The force-displacement profile of the probe is partitioned and then optimized sequentially, by immersing optimal trajectory design and iterative learning control into the Fibonacci search process. The proposed approach is illustrated through experimental implementations on two different types of polymer species, a polydimethylsiloxane sample, and a dental silicone sample, respectively.

Effect of Relative Humidity on the Viscoelasticity of Thin Organic Films Studied by Contact Thermal Noise AFM.

Material scientists are in need of experimental techniques that facilitate a quantitative mechanical characterization of mesoscale materials and, therefore, their rational design. An example is that of thin organic films, as their performance often relates to their ability to withstand use without damage. The mechanical characterization of thin films has benefited from the emergence of the atomic force microscope (AFM). In this regard, it is of relevance that most soft materials are not elastic but viscoelastic instead. While most AFM operation modes and analysis procedures are suitable for elasticity studies, the use of AFM for quantitative viscoelastic characterizations is still a challenge. This is now an emerging topic due to recent developments in contact resonance AFM. The aim of this work was to further explore the potential of this technique by investigating its sensitivity to viscoelastic changes induced by environmental parameters, specifically humidity. Here, we show that by means of this experimental approach, it was possible to quantitatively monitor the influence of humidity on the viscoelasticity of two different thin and hydrophobic polyurethane coatings representative of those typically used to protect materials from processes like weathering and wear. The technique was sensitive even to the transition between the antiplasticizing and plasticizing effects of ambient humidity. Moreover, we showed that this was possible without the need of externally exciting the AFM cantilever or the sample, i.e., just by monitoring the Brownian motion of cantilevers, which significantly facilitates the implementation of this technique in any AFM setup.

Effect of NaOCl and EDTA Solutions on Topography of ESX, TruShape and ProTaper Gold NiTi Rotary Instruments - An Atomic Force Microscopic Study.

Aim:The study was aimed to evaluate the effect of 5.25% NaOCl and 17% ethylenediaminetetraacetic acid (EDTA) Solutions on Surface Topography of ESX, TruShape, and ProTaper Gold nickel-titanium (NiTi) Rotary Instruments using Atomic Force Microscope (AFM).Materials and Methodology:A total of 27 each of three commercially available brands of endodontic NiTi instruments were analyzed, which were divided into three groups as follows: Group I: ESX (Brasseler, USA) Group II: Tru shape (Dentsply Tulsa, USA) Group III: Pro Taper Gold (Dentsply Tulsa, USA). These files were further divided into three subgroups containing nine files each, no immersion, immersion in 5.25% NaOCl for 5 min, and immersion in 17% EDTA for 5 min. Surface areas along 3 mm sections at the tip of the files (perfect squares of 10 μm × 10 μm) were analyzed using AFM operating in contact mode under ambient conditions.Statistical Analysis:Data were analyzed using two-way ANOVA and pairwise comparison of three main groups with respect to Ra and RMS by Tukey's multiple posthoc procedures.Results:Three-dimensional AFM images of the surface of all the rotary NiTi instruments, including new and those immersed in 5.25% NaOCl and 17% EDTA solutions, revealed topographic irregularities at the nanometric scale. Ra and RMS values of instruments treated with 5.25% NaOCl and 17% EDTA solutions were statistically higher than that of the new ones (P < 0.05).Conclusion:Using AFM for analysis indicated that short-term contact between 5.25% NaOCl and 17% EDTA solutions and NiTi instruments caused alterations in the topography of instruments.

Atomic Force Microscopy Local Oxidation of GeO Thin Films

Metastable germanium monoxide (GeO) thin-insulating films have been investigated as a new promising material for atomic force microscope (AFM) oxidation lithography. The kinetics of GeO layer oxidation performed in the tapping mode of AFM was found to have a logarithmic relationship to oxide height versus pulse duration. Effect of humidity on oxidation of germanium monoxide thin films was studied at relative humidity 40, 60, 80%. When local anodic oxidation of GeO layer was carried out by AFM operating in the contact mode in high voltage (≥9 V) regime and at high relative humidity (RH = 80%), the size and shape of fabricated oxide was changed drastically.

Artifacts and Practical Issues in Atomic Force Microscopy.

As with any other microscopic technique, in atomic force microscopy (AFM), problems can arise. Some of these happen due to improper use of the microscope by the operator, and some are due to particular characteristics of the sample. Some occur depending on the type of instrument, or from probe damage. Some of them are artifacts inherent in the technique. Knowledge of these issues is important for correct data acquisition and interpretation, and in many cases, training in AFM is inadequate. In this chapter we show examples of common artifacts in AFM and describe, where possible, how to overcome them. Other practical issues important for best practice in AFM operation, such as noise reduction and data processing, are also discussed.